We are utilizing two approaches to identify and clone genes involved in the control of early development in the mouse. Homeobox genes. Embryonic patterning in Drosophila is controlled by a regulatory gene network comprised, in part, of homeobox containing genes. The expression pattern of the murine homeobox (Hox) gene family suggests an analogous role in defining regional identity along the antero-posterior axis. However, Hox gene expression is not found in the anterior structures of the embryo, nor is it detected very early in development. To investigate the role of homeobox genes in these early events, we have cloned several divergent homeobox genes that are expressed in early development, and have studied their spatial expression pattern during embryogenesis by in situ hybridization. In the process, we have identified a new homeobox gene family in the mouse corresponding to the Distal-less (DII) gene of Drosophila, a gene involved in the patterning of the limbs and sensory organs of the fly. The murine Distal-less genes are the first homeobox genes described whose expression in the central nervous system (CNS) is restricted to forebrain. Thus these genes may contribute missing positional cues not provided by the previously identified vertebrate homeobox genes. In addition, the expression of another homeobox gene, prd-like, is the most anterior restricted of all known homeobox genes. Transcripts are seen very early in gastrulation, and become progressively restricted to Rathke's pouch, the rudiment of the anterior pituitary, indicating a potential role for the prd-like gene in the development of this organ. We plan conduct expression studies in embryonic stem (ES) cells and transgenic mice to investigate the developmental functions of these new homeobox genes. Insertional mutagenesis via Enhancer Trapping. To identify new genes involved in development we have introduced enhancerless/promoterless beta-galactosidase (lacZ) constructs into ES cells. Expression is only detectable if the construct integrates into an active gene region. We have screened enhancer trap cells for interesting patterns of lacZ expression in differentiating embryoid bodies and in chimeras, and have obtained several lines that differentially express the lacZ construct. One line shows restricted expression in the CNS of chimeras. The interrupted gene is being cloned via the molecularly distinguishable lacZ sequences. Breeding of chimeric mice obtained from these cells will potentially lead to the derivation of mouse mutants at these loci. The significance of this work lies in the potential to study the largely inaccessible period of early mammalian embryogenesis on the molecular level. This will ultimately allow a better understanding of both normal and abnormal development.